The soluble acyl-ACP delta9-desaturase (delta9D) from R. communis is a homodimeric enzyme in which each protomer contains a non-heme diiron active site capable of reductively activating oxygen during the regiospecific insertion of a cis double bond between C9 and C10 on stearoyl-ACP. Structurally, this enzyme is very similar to the small subunit of ribonucleotide reductase (R2), another metalloprotein within the class I non-heme diiron family. Previously, it has been suggested that the native activity of both enzymes proceed via 'half-sites' reactivity. Since 'half-sites' reactivity has been proposed for at least two members of this family, it is possible that this represents a common mechanistic motif for this class of enzymes. Therefore, the specific aim of this project is to directly determine if delta9D exhibits 'half-sites' activity by generating a heterodimeric enzyme which is catalytically functional on a single protomer only. This will be accomplished by co-expression of two distinct A9D genes; wild-type and an active site knock out mutant, on a single vector. Steady state kinetics for substrate and oxygen consumption can be determined for comparison to wild-type delta9D.